During the past year we have developed techniques for the immunohistochemical staining of prosthetic heart valves of both the mechanical and the biological type using multiple antibodies and fluorochromes. We have used these methods in conjunction with confocal microscopy and three-dimensional reconstructions of images obtained by Z-scanning of valvular surfaces, and we have demonstrated the occurrence of apoptosis in endothelial cells that had been specifically identified by labeling with antibodies to Factor VIII. In addition, we had been able to demonstrate the presence of matrix metalloproteinases in the connective tissues of bioprosthetic heart valves. We hope that methodology will also be useful in evaluating the surfaces of valves made of synthetic materials that this cannot be subjected to histological sectioning.